1. Field of the Invention
The present invention relates to a method for producing a transgenic pig suppressed in immune rejection response in which a gene coding for human HO-1 protein and a gene coding for TNFR1-Fc fusion protein are expressed simultaneously. More particularly, the present invention relates to a method for producing a transgenic pig suppressed in immune rejection response, comprising a) isolating somatic cells from a pig; b) introducing a gene coding for human HO-1 (Heme oxygenase-1) protein and a gene coding for human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion protein into the somatic cells; c) selecting and culturing the somatic cells that are introduced with the genes; d) removing the nucleus from an oocyte of the pig and fusing it with the selected somatic cell to prepare a somatic cell nuclear transferred embryo; and e) implanting the embryo. The present invention also relates to a transgenic pig suppressed in immune rejection response for organ transplantation that is produced by the above method, a somatic donor cell line for producing the transgenic pig, and a method for producing transplantable organ suppressed in immune rejection response from the transgenic pig.
2. Description of the Related Art
Pancreatic islet (hereinafter referred to as “islet”) transplantation is an innovative treatment strategy for diabetes. However, an explosive increase in the number of diabetic patients causes the severe lack of donor islets. Organ transplantation is the procedure of replacing diseased organs or parts of organs with healthy organs of another person when diseased organs lose their functions and drugs no longer help to treat the disease, and commonly includes transplantation of organs from living persons. Therefore, many attempts have been made to address the problem of the lack of donor islets due to the explosive increase in the number of diabetic patients. An example thereof includes stem cell xenotransplants, which is a therapeutic method of replacing damaged cells with stem cells that are differentiated and proliferated as much as needed. However, it has a limitation that the stem cells cannot be developed into an organ consisting of different types of cells. Therefore, xenotransplantation is used for direct replacement of organs when needed.
One of the promising alternative ways for human organs is islet xenotransplantation using animals capable of providing a sufficient quantity of donor islets, and many animals such as monkeys and pigs are attempted to be used to supply organs for xenotransplantation. Among them, pigs have numerous similarities with humans in terms of the anatomy and physiology, and their organs are similar in size to humans. In addition, pigs are easy to breed, and they have a short gestation period (112 days) and large litters (6-12 piglets). Owing to these advantages, the use of pig islet cells has been actively studied.
When islets are isolated for islet transplantation, they are cultured in vitro for a predetermined time prior to transplantation. However, if oxidative stress and other cell damages occur in islet cells during the isolation procedure, cell death is induced during in vitro cultivation of islet cells. Thus, the protection of insulin secreting cells from oxidative stress is thus regarded as a potential approach to prevent early graft failure of transplanted islets (Biarnes et al., Diabetes, 2002; 51: 66-72.). Because the inflammatory responses after islet transplantation might reduce a survival rate of implanted islet, it is also very important to control the inflammatory responses.
HO-1 (Heme oxygenase-1) is an enzyme that finally degrades heme into bilirubin and Fe2+, and is an antioxidant enzyme capable of cytoprotection via radical scavenging or apoptosis prevention.
TNF-α is a representative inflammatory cytokine, and a mediator that is mainly expressed in monocytes/macrophages and natural killer cells to induce inflammation. Many previous studies reported that TNF-α is one of the causes inducing immune rejection responses in xenotransplantation as well as allotransplantation (Carel J C. et al., Transplantation, 1993, 55(2):456-458; Rosenblum M G. et al., Cancer. Immunol. Immunother., 1995, 40(5):322-328; Lin Y. et al., Transplantation, 1997, 64(12):1677-1683; Lin H. et al., J. Surg. Res, 1997, 72(1):84-88; Jorgensen C. et al., Immunology, 1998, 93(4):518-523; Nagy T. et al., APMIS, 1999, 107(10):903-912; Benda B et al., Xenotransplantation, 2000, 7(3):206-213; Kirkiles-Smith N C. et al., J. Immunol., 2000, 164(12):6601-6609).
However, there was no technical idea that a gene coding for human HO-1 protein and a gene coding for TNFR1-Fc fusion protein capable of inhibiting human TNF-α are co-transfected into a pig to produce a transgenic pig, so as to protect islet cells from oxidative stress upon isolation and inflammatory responses after transplantation, thereby increasing the success rate of transplantation.
The present inventors have made many efforts to develop a method for suppressing immune rejection response and protecting islet cells from oxidative stress and inflammatory responses after transplantation. As a result, they have developed a porcine cell that is introduced with a gene coding for human HO-1 protein and a gene coding for TNFR1-Fc fusion protein composed of the extracellular region of human TNFR1 fused to IgG Fc region, and a method for producing a transgenic pig by nuclear transfer using the cell as a donor cell, thereby completing the present invention.